BAIKUNTHI DEVI KANYA MAHAVIDHYALAYA

BAIKUNTHI DEVI KANYA MAHAVIDHYALAYA. Submitted By :- Priyanka Kumari B.Sc. 3 Sem 59 Deptt. Of :- Zoology.

Contents. Introduction SDS Page Principle of SDS-PAGE Procedure Gel Casting Sample Preparation Gel Running Staining of Proteins Calculation of Rf Value Applications of SDS-PAGE Conclusion.

Introduction. Proteins are large, complex molecules that play critical roles in the body. Protein separation by electrophoresis is an important molecular technique. Polyacrylamide Gel Electrophoresis (PAGE) is used to separate proteins based on solubility, size, charge, and binding affinity. Acrylamide and BI acrylamide form cross-bridges to create the gel matrix..

What is SDS-PAGE?. SDS-PAGE (Sodium Dodecyl Sulphate - Polyacrylamide Gel Electrophoresis) is a denaturing technique used to separate proteins based on their molecular mass. • SDS denatures proteins and imparts a uniform negative charge. • Proteins are separated strictly by size. • Provides high resolution for complex mixtures. Native PAGE = non-denaturing separation..

Polyacrylamide Gel Electrophoresis PAGW. Native Page Proteins separated under non-denaturing conditions.

Principle of SDS-PAGE. • SDS binds to polypeptide chains, destroying protein structure. • SDS denatures proteins and imparts a negative charge. • In an electric field, proteins migrate towards the positive electrode. • Migration depends on molecular mass: smaller proteins migrate faster..

Gel Casting. Gel is cast using acrylamide, BI acrylamide, ammonium persulphate, and TEMED. Two parts: • Stacking gel: 5% acrylamide, large pores, aligns proteins. • Separating gel: 10–12% acrylamide, small pores, separates proteins..

Sample Preparation. • Proteins are treated with SDS and β-mercaptoethanol. • SDS denatures and coats proteins uniformly with negative charge. • β-mercaptoethanol breaks disulfide bonds. • Bromophenol blue is added as a tracking dye..

Gel Running. • The comb is removed, creating wells for loading samples. • Protein samples are loaded into wells using a micro syringe. • Electric field is applied; proteins migrate through gel matrix. • Bromophenol blue shows progress of electrophoresis..

Staining of Proteins. • After electrophoresis, proteins are invisible. • Stained with Coomassie blue or silver stain. • Destaining removes background, leaving visible protein bands..

Calculation of Rf Value. Relative mobility (Rf) = Distance migrated by protein ÷ Distance migrated by dye. • Used to determine molecular mass of proteins. • Protein standards (ladders) are used for comparison..

Applications of SDS-PAGE. • Estimation of molecular weight of proteins. • Analysis of protein purity. • Study of protein expression. • Identification of unknown proteins. • Used in zoology, biotechnology, and medicine..

Conclusion. SDS-PAGE is a powerful technique for separating proteins based on their molecular weight. It plays an important role in zoology, molecular biology, biotechnology, and medical research..

Thanks.